In vitro assay buffer for Cas9
Provided herein is a reaction mixture comprising Cas9 and a non-ionic surfactant, e.g., a polyoxyethylene surfactant. In certain embodiments, the reaction mixture may comprise a Cas9 protein, a guide RNA, a salt, a buffering agent, a nucleic acid target and a non-ionic surfactant. Kits are also provided. In certain embodiments, a kit may comprise: a Cas9 protein; and a concentrated reaction buffer comprising salt, a buffering agent and a non-ionic surfactant.
1. A reaction mixture for in vitro cleavage of a nucleic acid target comprising:
a) a Cas9 protein;
b) a guide RNA;
c) a salt;
d) a buffering agent;
e) a nucleic acid target; and
f) a non-ionic surfactant, wherein the non-ionic surfactant is a polyoxyethylene or polyoxypropylene surfactant, a glycosidic surfactant, or a bile salt surfactant;
wherein components a)-f) are combined in vitro.
2. The reaction mixture of claim 1 , wherein the buffering agent is selected from the group consisting of Tris, HEPES, TAPS, MOPS and MES.
3. The reaction mixture of claim 2 , wherein the buffering agent is at a concentration of 1 mM to 50 mM.
4. The reaction mixture of claim 1 , wherein the pH of the reaction mixture is in the range of pH 5 to 8.5.
5. The reaction mixture of claim 1 , wherein the reaction mixture comprises one or more of NaCl, MgCl 2 , MgSO 4 and KCl at a concentration in the range of 1 mM to 500 mM.
6. The reaction mixture of claim 5 , wherein the reaction mixture comprises NaCl at a concentration of 10 mM to 250 mM and MgCl 2 at a concentration of 1 mM to 50 mM.
7. The reaction mixture of claim 1 , wherein the presence of the surfactant increases the rate of cleavage of the nucleic acid target by at least 5%.
8. The reaction mixture of claim 1 , wherein the non-ionic surfactant is present in the reaction mixture at a concentration in the range of 0.005% to 1%.
9. The reaction mixture of claim 1 , wherein the non-ionic surfactant is a polyoxyethylene or polyoxypropylene surfactant.
10. The reaction mixture of claim 9 , wherein the polyoxyethylene surfactant is a Brij series surfactant.
11. The reaction mixture of claim 9 , wherein the polyoxyethylene surfactant is a Triton series surfactant.
12. The reaction mixture of claim 9 , wherein the polyoxyethylene surfactant is a Tween series surfactant.
13. The reaction mixture of claim 9 , wherein the polyoxyethylene surfactant is a Surfynol series surfactant.
14. The reaction mixture of claim 9 , wherein the polyoxyethylene surfactant is a Tergitol series surfactant.
15. The reaction mixture of claim 1 , wherein the non-ionic surfactant is a glycosidic surfactant.
16. The reaction mixture of claim 1 , wherein the non-ionic surfactant is a bile salt surfactant.
17. A reaction mixture for in vitro cleavage of a nucleic acid target comprising:
a) a Cas9 protein;
b) a guide RNA;
c) a salt;
d) a buffering agent;
e) a nucleic acid target; and
f) a non-ionic surfactant, wherein the non-ionic surfactant has an HLB in the range of 12-15;
wherein components a)-f) are combined in vitro.
18. The reaction mixture of claim 17 , wherein the non-ionic surfactant having an HLB in the range of 12-15 is selected from Triton X-100, Triton® SP-190, Triton XL-80N, Surfynol 465, Tergitol 15-S-7, 15-S-9, 15-S-12, Tergitol NP-9, Sulonic JL-80X, Ethofat 242/25, IGEPAL CA-720 and Lutensol NP 10.
19. A kit for preparing the reaction mixture of claim 1 , comprising:
a) a Cas9 protein; and
b) a concentrated reaction buffer comprising salt, a buffering agent and a non-ionic surfactant, wherein the non-ionic surfactant is a polyoxyethylene or polyoxypropylene surfactant, a glycosidic surfactant, or a bile salt surfactant.
20. A kit for preparing the reaction mixture of claim 17 , comprising:
a) a Cas9 protein; and
b) a concentrated reaction buffer comprising salt, a buffering agent and a non-ionic surfactant, wherein the non-ionic surfactant has an HLB in the range of 12-15.